Most biological functions are the result of interactions among molecules such as proteins, DNA, RNA and other small molecules. The large-scale analysis of proteins and their interactions, commonly referred to as proteomics, has become one of the most important disciplines for characterizing gene function, for building functional linkages between protein molecules, and for providing insight into the mechanisms of biological processes in a high-throughput mode.
A number of protein expression systems have been used as tools in biochemical research to analyze protein-protein interactions (e.g., antigen-antibody interactions and receptor-ligand binding). These expression systems include genetically engineered cell lines that over-express a protein of interest (e.g., receptor, antibody or enzyme) in modified bacteria, and phage display libraries of multiple proteins. Proteins prepared through these approaches can be isolated and either screened in solution or attached to a solid support for screening against a target of interest such as other proteins, receptor ligands, small molecules, and the like. Recently, a number of researchers have focused their efforts on the formation of arrays of proteins similar in concept to the nucleotide biochips currently being marketed. For example, WO 00/04389 and WO 00/04382 describe microarrays of proteins and protein-capture agents formed on a substrate having an organic thinfilm and a plurality of patches of proteins, or protein-capture agents. Also, WO 99/40434 describes a method of identifying antigen/antibody interactions using antibody arrays and identifying the antibody to which an antigen binds.
While arrays of proteins, and protein-capture agents provide a method of analysis distinct from nucleotide biochips, the preparation of such arrays requires purification of the proteins used to generate the array. Additionally, detection of a binding or catalytic event at a specific location requires either knowing the identification of the applied protein, or isolating the protein applied at that location of the array and determining its identity. Also, attachment of proteins to an array sometimes causes these proteins to lose their ability to interact with other proteins or ligands after immobilization.
What is needed is a means to identify protein binding events wherein a protein or portion thereof (e.g., bait polypeptide) is presented to a protein or portion thereof (e.g., prey polypeptide) in a way that provides a fast, high-throughput and reliable way to monitor protein-protein interactions and in which the proteins retain the ability to interact with other proteins. Additionally, it would be preferable to have the protein presented in a manner that allows for efficient isolation and/or identification of the proteins for which binding events are detected (e.g., identification of prey protein/polypeptide). Finally, the system should enable rapid analysis of the proteins by coupling of the arrays to detection systems that allow for the rapid, high-throughput analysis of chemical or biological samples. Such techniques would be valuable in identifying protein-protein interactions, subunits in multi-subunit complexes, as well as test compounds that may alter (e.g., enhance or inhibit) the protein-protein interaction.